A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

The purification of myosin long subfragment 2 by DEAE-Sepharose CL-6B ion-exchange chromatography

A new method for the purification of myosin long subfragment 2 is presented. This method is based on the fractionation, by DEAE-Sepharose CL-6B ion-exchange chromatography, of either chymotryptic hydrolysates of heavy meromyosin or tryptic hydrolysates of myosin total rod. Although emphasis is given to the purification of long subfragment 2, the method could be easily adapted to the purification of short subfragment 2.     

Non‐muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)

Non‐muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF‐alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365–1375, 2010. © 2010 Wiley‐Liss, Inc.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Design and synthesis of sulfonamidophenylethylamides as novel cardiac myosin activator

The sulfonamidophenylethylamide analogues were explored for finding novel and potent cardiac myosin activators. Among them, N-(4-(N,N-dimethylsulfamoyl)phenethyl-N-methyl-5-phenylpentanamide (13, CMA at 10 µM = 48.5%; FS = 26.21%; EF = 15.28%) and its isomer, 4-(4-(N,N-dimethylsulfamoyl)phenyl-N-methyl-N-(3-phenylpropyl)butanamide (27, CMA at 10 µM = 55.0%; FS = 24.69%; EF = 14.08%) proved to be efficient cardiac myosin activators both in in vitro and in vivo studies. Compounds 13 (88.2 + 3.1% at 5 µM) and 27 (46.5 + 2.8% at 5 µM) showed positive inotropic effect in isolated rat ventricular myocytes. The potent compounds 13 and 27 were highly selective for cardiac myosin over skeletal and smooth muscle myosin, and therefore these potent and selective amide derivatives could be considered a new class of cardiac myosin activators for the treatment of systolic heart failure.     

Septins regulate border cell surface geometry,shape, and motility downstream of Rho in Drosophila

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Molecular dynamics simulation of tropomyosin bound to actins/myosin in the closed and open states

Tropomyosin (Tpm) is a dimeric coiled-coil protein that binds to filamentous actin, and regulates actin-myosin interaction by moving between three positions corresponding to the blocked, closed, and open states. To elucidate how Tpm undergoes transitions between these functional states, we have built structural models and conducted extensive molecular dynamics simulations of the Tpm-actins/myosin complex in the closed and open states (total simulation time >1.4 μs). Based on the simulation trajectories, we have analyzed the dynamics and energetics of a truncated Tpm interacting with actins/myosin under the physiological conditions. Our simulations have shown distinct dynamics of four Tpm periods (P3-P6), featuring pronounced biased fluctuations of P4 and P5 toward the open position in the closed state, which is consistent with a conformational selection mechanism for Tpm-regulated myosin binding. Additionally, we have identified key residues of Tpm specifically binding to actins/myosin in the closed and open state. Some of them were validated as functionally important in comparison with past functional/clinical studies, and the rest will make promising targets for future mutational experiments.